Systems biology approach to dissect Salmonella-host interactions

Abstract

Systems understanding of the biological data is required to capture the complex biochemical information when a pathogen interacts with its host. Such interactions are dynamic and complex, especially because various components of the pathogen interact with the host at multiple levels. Understanding this complex data has been made somewhat feasible with the advent of systems biology. System biology involves studying the complex interactions which take place between genes, proteins, and other components in a biological network, compared to traditional biological research, where the focus is only on a small number of components. For instance, a systems biology study of host-pathogen interactions investigates the interaction between the components of two distinct organisms, a pathogen, and its animal host. The only way this is accomplished is through the integration and interpretation of the high-throughput data made available at various levels of detail. However, high-throughput techniques like sequencing and multi-omics not only generate a massive volume of data but also pose challenges to meaningfully extract and interpret the data. Salmonella sp. is a Gram-negative, intracellular pathogen and causes severe complications contributing significantly to the global burden of foodborne illnesses. Salmonella Typhi is a human-restricted pathogen that causes a systemic disease called Typhoid (enteric fever). On the other hand, Salmonella Typhimurium, a zoonotic pathogen, causes a less severe form of the disease called gastroenteritis in humans but a systemic typhoid-like disease in susceptible mice strains (typhoid model). The genomes of most organisms, including Salmonella, as they exist today are the result of millions of years of evolution - the acquisition or elimination of certain key genetic determinants, genomic rearrangements, and the insertion of novel genes into existing genetic circuitries. Reverse genetics has proven extremely useful in deciphering the function of such key genes and their protein products required during Salmonella pathogenesis. In the first part of this study, we sought to identify proteins that are exclusive to Salmonella sp. using comparative genome sequence analysis. Comparing the proteome of Salmonella with a non-redundant protein sequence database allowed us to not only detect sequences unique to the organism but also identify its conservation among the Salmonella serovars. We argue that such proteins with a unique sequence architecture might be acquired and evolutionarily retained in the genome of the organism only to serve some essential function. By careful genetic manipulation and phenotypic analysis, we were able to shed functional insights in a novel protein sequence, GtgF and demonstrate its role in S. Typhimurium adaptation in oxidative and genotoxic stress response mechanisms. Using a mouse typhoid model, the second part of our study assessed and compared the virulence function of GtgF in WT S. Typhimurium 14028s. In a susceptible mouse strain, like BALB/c, WT S. Typhimurium 14028s administered through the orogastric route is known to cause lethal infection. However, we observed that S. Typhimurium ΔgtgF was significantly more lethal (approx. two times) than the WT in an oral typhoid model. A marginally higher bacterial burden in tissues with increased inflammatory markers in the blood, and exacerbated tissue inflammation explained why the mice were more susceptible to infection with ΔgtgF strain. In addition to grvA, we believe gtgF might be an additional anti-virulence genetic determinant encoded by the same phage, Gifsy-2. Although it is unclear why bacterial pathogens possess anti-virulence genes and what evolutionary advantage they provide to their host, it is believed that certain intracellular pathogens evolve toward a less virulent form in order to attain a chronic carrier state, a phenomenon well-established in the intracellular pathogen, Salmonella. In the third and concluding part of the study, we developed a drug-repurposing strategy to design antibiotic adjuvants that can target the MsgA-like family of proteins in Salmonella. As these proteins are mostly conserved in pathogenic organisms, targeting this family of proteins has the selective advantage of having negligible and unintentional anti-commensal activity. It is well-known that targeting conditionally essential proteins can impede the rate of anti-microbial resistance. While discovering novel drugs or even new classes of antimicrobials might be an extremely time-consuming and resource-intensive affair, orthogonal approaches such as designing antibiotic potentiators can be implemented simultaneously to preserve our existing arsenal of antimicrobials. Through high-throughput virtual screening with an FDA-approved drug subset, we identified potential drugs which can be repurposed as antibiotic adjuvants against the intended targets. The computational findings were validated to determine whether these drugs can indeed potentiate the action of the co-administered antibiotic. Some true validations obtained in this study clearly demonstrate that the proposed methodology holds promise; however, it remains to be seen whether these observations hold true for drug-resistant isolates. Consequently, the mechanism of action of the drugs must also be established. However, as a proposed methodology, these observations has significant ramifications in the antibiotic resistance problem. In summary, the entire workflow presented here is extremely generic and can be adapted for any pathogenic microorganism to tackle the rising concern of anti-microbial resistance.